Tyrosine hydroxylase (TH) has been purified to homogeneity from cultured PC12 cells. The enzyme has a subunit molecular weight of 58,800. Two dimensional electrophoresis of the purified enzyme revealled three isozymic forms differing in charge (pI between 5.3-5.6) and which were positively identified by Western immunoblots. Amino acid analysis indicated a high content of hydrophobic amino acids which was confirmed by the observation that the enzyme also binds tightly to phenylsepharose, a hydrophobic matrix. Antibodies against PC12 TH were raised in rabbits and the antibody was highly specific. Blots of crude protein preparations from brain adrenal chromaffin cells, rat striatum, and cultured PC12 cells onto nitrocellulose paper revealed that the antibody recognized a single protein of Mr 58,800 in each tissue which corresponds to the Mr of purified TH. The anti-TH also immunoprecipitated TH enzyme activity from each tissue but interaction of the antibody with TH neither activated nor inhibited enzyme activity as such. The anti-TH does not cross react with other monooxygenase enzymes including tryptophan hydroxylase and phenylalanine hydroxylase. The purified enzyme is a good substrate for various protein kinase enzymes. The enzyme shows little activity toward tryptophan as a substrate and expressed no phenylalanine hydroxylase activity whatsoever.